Hordein accumulation in developing barley grains

The temporal pattern of accumulation of hordein storage proteins in developing barley grains was studied by enzyme-linked immunosorbent assay (ELISA), western blot and liquid chromatography tandem mass spectrometry (LC-MS/MS). Hordein accumulation was compared to the pattern seen for two abundant control proteins, serpin Z4 (an early accumulator) and lipid transferase protein (LTP1, a late accumulator). Hordeins were detected from 6 days post-anthesis (DPA) and peaked at 30 DPA. Changes in fresh weight indicate that desiccation begins at 20 DPA and by 37 DPA fresh weight had decreased by 35%. ELISA analysis of hordein content, expressed on a protein basis, increased to a maximum at 30 DPA followed by a 17% decrease by 37 DPA. The accumulation of 39 tryptic and 29 chymotryptic hordein peptides representing all classes of hordein was studied by LC-MS/MS. Most peptides increased to a maximum at 30 DPA, and either remained at the maximum or did not decrease significantly. Only five tryptic peptides, members of the related B1- and γ1-hordeins decreased significantly by 21–51% at 37 DPA. Thus, the concentration of some specific peptides was reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. In addition to a suite of previously characterized hordeins, two novel barley B-hordein isoforms mapping to wheat low molecular weight glutenins (LMW-GS-like B-hordeins), and two avenin-like proteins (ALPs) sharing homology with wheat ALPs, were identified. These identified isoforms have not previously been mapped in the barley genome. Cereal storage proteins provide significant nutritional content for human consumption and seed germination. In barley, the bulk of the storage proteins comprise the hordein family and the final hordein concentration affects the quality of baked and brewed products. It is therefore important to study the accumulation of hordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food processing properties

Hordein Accumulation in Developing Barley Grains

The temporal pattern of accumulation of hordein storage proteins in developing barley grains was studied by enzyme-linked immunosorbent assay (ELISA), western blot and liquid chromatography tandem mass spectrometry (LC-MS/MS). Hordein accumulation was compared to the pattern seen for two abundant control proteins, serpin Z4 (an early accumulator) and lipid transferase protein (LTP1, a late accumulator). Hordeins were detected from 6 days post-anthesis (DPA) and peaked at 30 DPA. Changes in fresh weight indicate that desiccation begins at 20 DPA and by 37 DPA fresh weight had decreased by 35%. ELISA analysis of hordein content, expressed on a protein basis, increased to a maximum at 30 DPA followed by a 17% decrease by 37 DPA. The accumulation of 39 tryptic and 29 chymotryptic hordein peptides representing all classes of hordein was studied by LC-MS/MS. Most peptides increased to a maximum at 30 DPA, and either remained at the maximum or did not decrease significantly. Only five tryptic peptides, members of the related B1- and γ1-hordeins decreased significantly by 21–51% at 37 DPA. Thus, the concentration of some specific peptides was reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. In addition to a suite of previously characterized hordeins, two novel barley B-hordein isoforms mapping to wheat low molecular weight glutenins (LMW-GS-like B-hordeins), and two avenin-like proteins (ALPs) sharing homology with wheat ALPs, were identified. These identified isoforms have not previously been mapped in the barley genome. Cereal storage proteins provide significant nutritional content for human consumption and seed germination. In barley, the bulk of the storage proteins comprise the hordein family and the final hordein concentration affects the quality of baked and brewed products. It is therefore important to study the accumulation of hordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food processing properties

Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference

<div><p>Background</p><p>Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed.</p> <p>Aim</p><p>The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined.</p> <p>Results</p><p>A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation.</p> <p>Conclusions</p><p>Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.</p> </div

Hordeins do not bleed off in aqueous washes.

<p>Duplicate 50 mg aliquots of Sloop flour were extracted in 1 mL of 50% IPA containing 1% (w/v) DTT (IPA/DTT) and an aliquot containing 20 µg protein dried in a SpeedyVac, dissolved in Urea/SDS and resolved on duplicate SDS-PAGE gels (lanes P) which were either stained in colloidal Coomassie Blue (A and C) or blotted to nitrocellulose (iBLOT Promega), blocked in PBST containing 5% skim milk powder, 1% (w/v) Tween overnight at 4°C. The blot was exposed to anti-gliadin-HRP (Sigma) diluted at 1/2000 for 30 min, then washed in PBST, and the signal produced by ECL reagent (Amersham) exposed to Hyperfilm (Amersham) (B and D). Similarly, 50 mg of duplicate flour samples were first washed in 1 mL of water, and centrifuged to give a supernatant and a pellet, which was dissolved in IPA/DTT. Aliquots containing 20 µg protein from both pellet (P1) and supernatant (S1) were dried, redissolved and subject to SDS-PAGE as above. The process was repeated with additional aqueous washing steps to produce supernatants S2–4 and pellets P2–4. Low molecular weight proteins were removed from pellets (A: arrowed) and extracted into supernatants (C: arrowed) after 2 washes. Proteins were calibrated with Benchmark protein ladder (St; Invitrogen).</p

The effect of DTT, hydrogen peroxide and urea on the ELISA response.

<p>The response of ELISA Systems sandwich assay containing total Sloop hordein (500 ppb) and either (A) DTT, or H₂O₂ or (B) urea, diluted in ED buffer and added to the ELISA wells at the concentration indicated above and processed as described.</p

SDS-PAGE of hordein fractions corresponding to the protein peaks in <b>Fig 9A, B, and C</b> were pooled as indicated, re-chromatographed, lyophilised, and 20 µg of each fraction examined by SDS-PAGE and stained with Coomassie G250.

<p>Lane 1, total hordein from cv Sloop; 2, did not contain any protein; 3, C-hordein from cv Sloop; 4, B-hordein from cv Sloop; 5, γ-1-, -2-hordeins from cv Sloop; 6, γ-1-, -2-, -3-hordeins from cv Sloop; 7, total hordein from Risø 56; 8, C-hordein from Risø 56; 9, γ-1, -2, -3 hordeins from Risø 56. A faint D-hordein band was seen in lane 7. Proteins were compared to standard proteins (M; Benchmark Protein Ladder, Invitrogen).</p

Repeated freeze thaw cycles do not affect the ELISA response.

<p>One mL aliquots of either total Sloop hordein (1.0 mg/mL; in a solution containing 8 M urea, 1% (w/v) DTT, 20 mM triethanolamine-HCl adjusted to pH 6 (A) or Sloop beer (B) were repeatedly frozen in liquid nitrogen and thawed in a water bath at RT. Aliquots were retained at 4°C after the indicated freeze-thaw cycle and diluted with ED buffer as required and duplicates analysed for hordeins by an ELISA Systems kit. The mean A450±SE is shown. Error bars are not shown where the S.E. is less than the line thickness. In each experiment the means were not significantly different (P<0.05) by one-way ANOVA (GenStat) and the least significant difference (LSD) is shown. Final concentration of hordein was 1000 ppb. Final dilution of Sloop beer was 1/500.</p

Comparison of hordein in flour and beer samples by <i>ELISA Systems</i> sandwich ELISA kit.

1<p>The <i>ELISA Systems</i> data is reproduced from (Tanner et al, this volume, this journal) with permission.</p><p>Hordein in flour and beer were calibrated against an appropriate hordein standard prepared from the respective flour.</p

Optimisation of dilution and extraction protocol for ELISA Systems assay.

<p>The response of the ELISA Systems sandwich assays to a commercial lager, (Tanner this journal, this issue, Beer 7) extracted with different buffers was measured by adding triplicate samples of commercial beer (100 µL) to 0.9 mL of either: (A & B) dilute ELISA Systems extraction buffer; (C, D, and E) <i>Ridascreen</i> extraction cocktail; (F) 60% (v/v) ethanol; or (G) Urea/DTT. The solutions were mixed at either room temperature (A, C, D, F, and G) or 60°C (B, E) for 1 h and an aliquot diluted 1/100 fold with ED buffer (A, B, D, F, G). Solutions C and E were diluted with ten-fold with 80% (v/v) ethanol, and then diluted a further ten-fold with dilute RD buffer and assayed for hordein. Hordeins were measured by adding duplicate 50 µL aliquots to 50 µL of ED buffer in ELISA Systems wells and assayed as described. The mean A450±SE is shown. The maximum concentrations of urea and DTT in assays of solution G were 40 mM and 0.3 mM respectively. The untransformed data were analysed by one-way ANOVA and the LSD is shown. Columns with the same letter were not significantly different (GenStat).</p